The ability to genetically manipulate insect disease vectors such as the malaria vector mosquito Anopheles gambiae remains rudimentary relative to the abundance of molecular tools that are currently available in model insects such as Drosophila. Recently, artificial nucleases have allowed targeted genome editing in species, such as the zebrafish and rat, that previously lacked effective tools. These chimeric nucleases combine a programmable sequence-specific DNA-binding domain with a non-specific nuclease domain to generate a double strand break at a desired genomic locus, which when imprecisely repaired can result in gene inactivation (knockouts). If these lesions are generated within the embryonic germline the propagation of targeted mutant alleles is possible. In order to enable this approach for Anopheles gambiae, we will design and optimize a series of custom nucleases targeting a pair of well-characterized odorant receptor (AgOr) genes that play essential roles in olfactory signal transduction. In these studies, we will compare two different programmable nuclease platforms for their efficiency in promoting gene inactivation in the Anopheles germline, with the goal of establishing a robust reverse genetic approach for this organism. The utility of this strategy will be demonstrated by evaluating the effect of various AgOr knockouts on chemosensory responses in adult and larval stage mosquitoes. In addition to advancing our basic knowledge of chemosensory signal transduction in this important disease vector, the proposed studies, if successful, should provide a basis for the laboratory- based application of these and related gene modification tools in Anopheles and a wide range of related vector species.